Indels and

Structural variants

Learning objectives

1. Understand the different types of structural variants

2. Learn the different algorithms to detect structural variants in

each sequencing technology

3. Annotate structural variants in VCF format

4. Visualize structural variants in IGV

5. Merge structural variants in VCF format

6. Benchmark structural variants

Types of genetic variation

ctctgag

ctccgag

Single-nucleotide

polymorphisms

(SNPs)

Single-nucleotide

variants

(SNVs)

Types of genetic variation

ctctgag

ctccgag

Single-nucleotide

polymorphisms

(SNPs)

ctctgag

ctc--ag

Insertion-deletion

polymorphisms

(INDELs)

Single-nucleotide

variants

(SNVs)

Types of genetic variation

ctctgag

ctccgag

Single-nucleotide

polymorphisms

(SNPs)

ctctgag

ctc--ag

Insertion-deletion

polymorphisms

(INDELs)

ctc ag

ctcaag

Structural

variants

(SVs)

Single-nucleotide

variants

(SNVs)

Types of genetic variation

ctctgag

ctc--ag

Insertion-deletion

polymorphisms

(INDELs)

ctc ag

ctcaag

Structural

variants

(SVs)

Differences between Indels and SVs are often blurry. In

general:

Biology

• Indels – Replication slippage

• SVs

• Recombination issues: Nonallelic homologous recombination

• DNA double strand break repair: Non-homologous

end-joining (NHEJ)

Size (historical alignment of short-reads)

• Indels < 50bp

• SVs > 50bp

Detection

• Indels – Gaps and insertions in the alignment process,

detected by variant callers

• SVs – Specialized tools to detect signals of SV (although

long read mappers can produce large gaps)

Indels

• In(sertion)Del(eletion)

• A mutation that results from the gain or loss of a sequence

Reference TCCAGCAATCAGCGTCAAGCTT

Sample TCAAGCAA---GCGTCAAGCAA

Reference TCCAGCAA GCGTCAAGCTT

Sample TCAAGCAA(TCA)GCGTCAAGCAA

Indels

• Replication slippage

Indels

• Replication slippage

Indels

• In(sertion)Del(eletion)

Indels

• Left alignment of the variants (bcftools norm)

Indel realignment

• Local realignment around indels

• Correct mapping errors – more precise indel discovery

Indel realignment

• Local realignment around indels

• Correct mapping errors – more precise indel discovery

Indel realignment

Before

After

Indel realignment

Before

After

Indel realignment

• GATK

Indel realignment

• ABRA

java -Xmx16G -jar $abra_jar

--in <bam_file>

--out <bam_file>

--ref <reference_genome>

--threads <n>

--tmpdir <dir>

> <file>.log

Structural variants

B CA

Reference

DB CA B

Duplication

Inversion

Deletion

DB CX

Insertion

Translocation

Structural variants

Nonallelic homologous recombination

Structural variants

Nonallelic homologous recombination

Double strand break

Non-

homologous

end-joining

(NHEJ)

1 2 3

1 3

1 2 3

1 3

Homologous

recombination

(HR)

Structural variants

Ref.

Exp.

(a) Depth of

coverage

(b) Paired-end

mapping

(d) de novo

assembly

Low High

Resolution

Evidence of structural differences

Structural variants

Structural variants - Deletions

Depth of Coverage

Drop in coverage

is a sign of a

deletion

DNA or

cDNA

fragment

read from

each end

align to

reference

Structural variants – Paired end sequencing

DNA or

cDNA

fragment

read from

each end

align to

reference

Structural variants – Paired end sequencing

DNA or

cDNA

fragment

read from

each end

align to

reference

R F R

F R

Structural variants – Paired end sequencing

DNA or

cDNA

fragment

read from

each end

insert siz e (or template length)

align to

reference

inferred ins ert size (or observed template length)

Structural variants – Paired end sequencing

reference

genome

subject

Structural variants – Deletions

Insert size

reference

genome

subject

A B

Structural variants – Deletions

Insert size

reference

genome

subject

A B

Structural variants – Deletions

Insert size

reference

genome

subject

A B

Structural variants – Deletions

Insert size

reference

genome

subject

A B

Structural variants – Deletions

Insert size

reference

genome

subject

A B

Structural variants – Deletions

Insert size

reference

genome

subject

A B

Structural variants – Deletions

Insert size

reference

genome

subject

Inferred insert size

Expected insert siz e

Inferred insert size is greater than expected value

Insert size

Structural variants – Deletions

reference

genome

subject

Pairs with larger than expected insert size are colored red in IGV

Structural variants – Deletions

Insert size

Structural variants – Deletions

Insert size

Long insert

size

reference

genome

subject

Reads spanning the breakpoints will get split when

mapped to the reference genome and mapped to both

sides of the breakpoints – precise breakpoint detection

Structural variants – Deletions

Split reads

reference

genome

subject

1. Depth of coverage

Structural variants – Deletions

reference

genome

subject

1. Depth of coverage

Structural variants – Deletions

reference

genome

subject

1. Depth of coverage

2. Paired-end mapping

Structural variants – Deletions

reference

genome

subject

1. Depth of coverage

2. Paired-end mapping

Structural variants – Deletions

reference

genome

subject

1. Depth of coverage

2. Paired-end mapping

3. Split read

Structural variants – Deletion

reference

genome

subject

Structural variants – Deletion

1. Depth of coverage

2. Paired-end mapping

3. Split read

reference

genome

subject

Structural variants – Insertion

1. Paired-end mapping

reference

genome

subject

Structural variants – Insertion

1. Paired-end mapping

Shorter insert

size than

expected is a

sign of an

insertion

reference

genome

subject

Structural variants – Insertion

1. Paired-end mapping

2. Split read

reference

genome

subject

Structural variants – Insertion

1. Paired-end mapping

2. Split read

Split reads with

an unmapped

region is a sign

of a read

spanning an

insertion

reference

genome

subject

1. Coverage

Structural variants - Duplication

reference

genome

subject

1. Coverage

Increase in

coverage is a

sign of a

duplication

Structural variants - Duplication

Normal

Tumor

Duplication

Structural variants – Duplication

reference

genome

subject

Structural variants – Duplication

1. Coverage

2. Split read

Split reads with

overlapping

sequences is a

sign of a tandem

duplication

Orientation of paired reads can reveal

evidence of structural events, including:

• Inversions

• Duplications

• Translocations

Structural variants

A B

reference

genome

subject

Structural variants - Inversions

reference

genome

subject

A B

Structural variants - Inversions

reference

genome

subject

A B

Structural variants - Inversions

reference

genome

subject

A B

Structural variants - Inversions

reference

genome

subject

A B

Structural variants - Inversions

reference

genome

subject

A B

Structural variants - Inversions

reference

genome

subject

forward forward

A B

Structural variants - Inversions

reference

genome

subject

A B

Structural variants - Inversions

reference

genome

subject

reverse

A B

Structural variants - Inversions

reference

genome

subject

A B

Structural variants - Inversions

Paired reads

with the same

orientation

indicate the

breakpoint of an

inversion

Pairs with same

reverse orientation

Pairs with same

forward

orientation

Note drop in coverage at breakpoints

Structural variants - Inversions

Aaron Wenger , PacBio

Pacbio

Illumina

Structural variants - Inversions

Long reads can

span long

deletions without

splitting the read!

They will show

long gaps

A B

A C

A B C

A B

concordant (+/-)

too big (+/-)

= deletion

Test

genome

Ref.

genome

too small (+/-)

= spanned insertion

A C

everted (-/+)

= tandem duplication

BA C

same strand (+/+ or -/-)

= inversion

Quinlan and Hall, 2012

Structural variants

Quinlan and Hall, 2012

Paired-end

Sample

Ref.

Split-read

Structural variants

Structural variant annotation

CGTGTtgtagtaCCGTAA Reference

CGTGT-------CCGTAA Sample

CGTGTtgtagtaCCGTAA Reference

CGTGT-------CCGTAA Sample

1. Direct sequence notation

#CHROM

POS

REF

ALT

QUAL

FILTER

INFO

FORMAT

SAMPLE

chr1

TTGTAGTA

PASS

…

1/1

Structural variant annotation

CGTGTtgtagtaCCGTAA Reference

CGTGT-------CCGTAA Sample

1. Direct sequence notation

#CHROM

POS

REF

ALT

QUAL

FILTER

INFO

FORMAT

SAMPLE

chr1

TTGTAGTA

PASS

…

1/1

2. Symbolic notation

#CHROM

POS

REF

ALT

QUAL

FILTER

INFO

FORMAT

SAMPLE

chr1

<DEL>

PASS

…

1/1

Structural variant annotation

CGTGTtgtagtaCCGTAA Reference

CGTGT-------CCGTAA Sample

1. Direct sequence notation

#CHROM

POS

REF

ALT

QUAL

FILTER

INFO

FORMAT

SAMPLE

chr1

TTGTAGTA

PASS

…

1/1

2. Symbolic notation

#CHROM

POS

REF

ALT

QUAL

FILTER

INFO

FORMAT

SAMPLE

chr1

<DEL>

PASS

SVLEN=7

1/1

Structural variant annotation

Assignment 2

We will compare Indel and SV calling for three sequencing

technologies:

1. Illumina

2. PacBio

3. Nanopore

The benchmarking will be done using the well-characterized

HG002

Assignment 2

Chromosome

Position

Reference

Alternate

Quality

Filter

Info

Format

Genotype

10415

ACCCTAACCCTAACCCTAACCCTAAC

…

1/1

62297

TCTTC

…

1/1

Assignment 2 – Intersecting SV

Chromosome

Position

Reference

Alternate

Quality

Filter

Info

Format

Genotype

10415

ACCCTAACCCTAACCCTAACCCTAAC

…

1/1

62297

TCTTC

…

1/1

10414

10440

62296

62297

Step 1: Get VCF file to BED file

VCF

BED

Chromosome

Position

Reference

Alternate

Quality

Filter

Info

Format

Genotype

10415

ACCCTAACCCTAACCCTAACCCTAAC

…

1/1

62297

TCTTC

…

1/1

10414

10440

62296

62297

Step 2: Get benchmark VCF file to BED file

VCF

BED

Assignment 2 – Intersecting SV

Step 3: Intersect coordinates of SV

Assignment 2 – Intersecting SV

Step 3: Intersect coordinates of SV

Percent overlap

If percent overlap > x% -> Same SV

Assignment 2 – Intersecting SV

Step 3: Intersect coordinates of SV

Percent overlap

If percent overlap > x% -> Same SV

* Reciprocal overlap – Both Variants need to overlap with each other

Assignment 2 – Intersecting SV

Step 3: Intersect coordinates of SV

30 55

Percent overlap

If percent overlap > x% -> Same SV

* Reciprocal overlap – Both Variants need to overlap with each other

Assignment 2 – Intersecting SV

Step 3: Intersect coordinates of SV

30 55

Percent overlap

If percent overlap > x% -> Same SV

* Reciprocal overlap – Both Variants need to overlap with each other

bedtools intersect

-a sample.bed

-b benchmark.bed

-f 0.50

-r

Assignment 2 – Intersecting SV

Filtering SV by length

• Most structural variant callers will add an INFO/SVLEN field that

can be used for filtering SVs by length

Filtering Indels by length

• BCFtools can be used to easily filter Indels by length using ”ILEN”

• ILEN will count up the length of the indel for you: positive numbers

are for insertions, and negative are deletions.

# To remove indels smaller than 5bp:

bcftools view -i '(ILEN >= -5 && ILEN <= 5)’

TYPE is a built-in that will determine whether a variant is an indel, snp,

etc. on-the-fly, so you can use the OR logic to include variants that

aren't indels (i.e. snps) using the second command. Note that

otherwise, ILEN explicitly excludes anything that's not an indel.

# To remove indels smaller than 5bp and preserve SNPs

Bcftools view -i '(ILEN >= -5 && ILEN <= 5) || TYPE!="INDEL”’